&y^ 


\S 


I  April  2.(i,  1914. 

[  AGRICULTURAL  EXPERIMENT  STATION, 

WILCOX,  Special  Agent  in  Charge. 


££« 


Bulletin  No.  33. 


THE  ORGANIC  NITROGEN  OF 
HAWAIIAN  SOILS. 


BY 


W.  P.  KELLEY, 

Chemist, 


AND 


R.  THOMPSON, 

Assistant  Chemist. 


UNDER  THE  SUPERVISION  OF 
OFFICE    OF    EXPERIMENT    STATIONS, 

U.  8.   DEPARTMENT  OF  AGRICULTUR 


vS.^'*;/ 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1914. 


/"' 


r 


Issued  April  25,  1914. 

HAWAII  AGRICULTURAL  EXPERIMENT  STATION, 

E.  V.  WILCOX,  Special  Agent  in  Charge. 


Bulletin  No.  33. 


THE  ORGANIC  NITROGEN  OF 
HAWAIIAN  SOILS. 


BY 

W.  P.  KELLEY, 

Chemist, 

AND 

ALICE  R.  THOMPSON, 

Assistant  Chemist. 


VNI-ER  THE  SUPERVISION  OF 
OFFICE    OF    EXPERIMENT    STATIONS, 

T.   S.    DEPARTMENT  OF   AGRICULTURE. 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1914. 


HAWAII    AGRICULTURAL    EXPERIMENT    STATION,    HONOLULU. 

[Under  the  supervision  of  A.  C.  True,  Director  of  the  Office  of  Experiment  Stations,  United  States  De- 
partment of  Agriculture.] 

Walter  H.  Evans,  Chief  of  Division  of  Insular  Stations,  Office  of  Experiment  Stations. 

STATION   STAFF. 

E.  V.  Wilcox,  Special  Agent  in  Charge. 
J.  Edgar  Higgins,  Horticulturist. 

W.  P.  Kelley,  Chemist. 

C.  K.  McClelland,  Agronomist. 

D.  T.  Fullaway,  Entomologist. 

W.  T.  McGeorge,  Assistant  Chemist. 
Alice  R.  Thompson,  Assistant  Chemist. 
C.  J.  Hunn,  Assistant  Horticulturist. 
V.  S.  Holt,  Assistant  in  Horticulture. 
€.  A.  Sahr,  Assistant  in  Agronomy. 

F.  A.  Clowes,  Superintendent  Hawaii  Substations. 
W.  A.  Anderson,  Superintendent  Rubber  Substation. 
J.  de  C.  Jerves,  Superintendent  Homestead  Substation. 
Joseph  K.  Clark,  Superintendent  Waipio  Substation. 
George  Copp,  Superintendent  Kula  Substation. 

(2) 


LETTER  OF  TRANSMITTAL 


Honolulu,  Hawaii,  August  4,  1913. 
Sir:  I  have  the  honor  to  submit  herewith,  and  to  recommend  for 
publication  as  Bulletin  33  of  the  Hawaii  Experiment  Station,  a  paper 
on  The  Organic  Nitrogen  of  Hawaiian  Soils,  by  W.  P.  Kelley,  chemist, 
and  Alice  R.  Thompson,  assistant  chemist.  On  account  of  the  great 
importance  of  nitrogen  to  growing  plants  it  is  highly  desirable  to  know 
more  about  the  nature  of  the  nitrogenous  substances  in  soils.  In  the 
research  which  served  as  a  basis  for  the  present  bulletin  an  attempt 
was  made  to  determine,  so  far  as  possible,  the  forms  in  which  nitrogen 
occurs  in  the  soils,  and  the  relative  percentages  of  the  various  nitrog- 
enous products.  These  studies  furnish  an  important  contribution  to 
the  subject,  which  will  later  be  supplemented  by  a  study  of  the 
products  of  various  vegetable  proteids  when  acted  upon  by  bacteria. 
Respectfully, 

E.  V.  Wilcox, 
Special  Agent  in  Charge. 
Dr.  A.  C.  True, 

Director  Office  of  Experiment  Stations, 

U.  S.  Department  of  Agriculture,   Washington,  D.  C. 

Publication  recommended. 
A.  C.  True,  Director. 

Publication  authorized. 

D.  F.  Houston,  Secretary  of  Agriculture. 

(3) 


CONTENTS. 

Page. 

Introduction 5 

The  nitrogen  of  Hawaiian  soils 7 

Nitrate  and  ammonia 7 

Organic  nitrogen 8 

Amids 9 

Basic  nitrogen 10 

Nonbasic  nitrogen 11 

Effects  of  aeration  on  soil  nitrogen 11 

Humus  nitrogen 12 

Nitrogen  dissolved  in  the  preliminary  1  per  cent  hydrochloric  acid  extrac- 
tion   13 

Separation  of  different  forms  of  nitrogen  in  humus 14 

A.mid  nitrogen 15 

Basic  nitrogen 16 

Nonbasic  nitrogen 17 

Determination  of  humus  nitrogen 19 

Percentage  of  nitrogen  in  humus  of  Hawaiian  soils 20 

Summary 21 

(4) 


THE  ORGANIC  NITROGEN  OF  HAWAIIAN  SOILS. 


INTRODUCTION. 

The  greater  part  of  soil  nitrogen  may  reasonably  be  assumed  to  have 
been  bound  up  at  one  time  or  another  in  protein  combinations,  since 
the  nitrogen  in  the  main  has  been  derived  from  vegetable  sources. 
Limited  amounts  of  other  nitrogen  bodies,  such  as  alkaloids,  etc., 
also  find  their  way  into  soils,  but  the  nitrogen  from  such  compounds 
could  hardly  be  expected  to  amount  to  more  than  a  small  percentage 
of  the  total  nitrogen  present.  The  chemistry  of  soil  nitrogen  and  the 
changes  that  it  undergoes,  therefore,  must  be  largely  those  of  plant 
proteins,  brought  about  under  complex  and  indeed  extremely  varia- 
ble conditions.  A  great  host  of  organisms  inhabiting  soils  are  asso- 
ciated with  the  transformations  of  the  organic  nitrogen  bodies,  and 
the  conditions  and  environment  in  which  the  organisms  function 
not  only  materially  alter  the  rates  of  their  action  but  also  determine 
largely  what  the  end  products  shall  be.  The  presence  of  various 
chemical  substances,  both  organic  and  inorganic,  the  acidity  or 
alkalinity,  and  the  degree  of  porosity  of  the  soil,  all  exert  important 
influences  on  the  activity  of  soil  organisms. 

During  the  past  few  years  considerable  study  has  been  devoted 
to  the  nitrogen  compounds  of  the  soil.  In  1905-6,  Shorey,1  while 
chemist  at  this  station,  applied  to  a  coffee  soil  from  the  island  of 
Hawaii  the  methods  formerly  used  in  the  study  of  protein,  and  thus 
determined  the  amounts  of  basic,  nonbasic,  ammonia  nitrogen,  etc., 
split  off  by  means  of  boiling  acids.  In  connection  with  his  studies 
a  pyridin  derivative,  picolin  carboxylic  acid,  was  isolated  and  identi- 
fied, this  being  the  first  definite  organic  nitrogen  compound  to  be 
isolated  from  a  soil.  Recently  a  number  of  other  studies  on  soil 
nitrogen  have  been  reported.2 

The  researches  previously  made  on  this  subject  naturally  divide 
themselves  into  two  classes,  as  indicated  by  the  work  of  Shorey. 
First,  a  study  of  the  individual  compounds  that  occur  in  natural 
soils;  second,  a  study  of  the  products  formed  by  acid  hydrolysis. 
The  extensive  researches  of  Schreiner,  Shorey,3  and  their  associates 

i  Hawaii  Sta.  Rpt.  1906,  pp.  87-89. 

»  Jodidi,  Michigan  Sta.  Tech.  Bui.  4  (1909);  Iowa  Sta.  Research  Buls.  land.3  (1911);  Robinson,  Michigan 
Sta.  Tech.  Bui.  7(1911);  Lathrop  and  Brown,  Pennsylvania  Sta.  Rpt.  1910,  pp.  118-120;  Jour.  Indus,  and 
En-in.  (hem.,  3  (1911),  pp.  657-660. 

»  U.  S.  Dept.  Agr.,  Bur.  Soils  Bute.  47,  S3,  71,  B0,  87,  8! 

C5) 


in  the  Bureau  of  Soils,  conducted  mainly  with  reference  to  the  indi- 
vidual compounds  actually  occurring  in  soils,  have  brought  to  light 
much  important  information.  Likewise  the  work  of  Jodidi,1  Robin- 
son,1 and  others,  is  of  interest  in  its  bearing  on  the  hydrolytic  prod- 
ucts split  off  by  means  of  acids.  From  the  investigations  on  the 
hydrolytic  products  it  has  been  shown  that  soils  vary  considerably 
in  regard  to  the  relative  percentages  of  the  several  groups  of  nitrogen 
compounds  split  off  in  the  treatment,  and  although  only  a  compara- 
tively small  number  of  soils  have  been  studied,  these,  in  the  main, 
have  been  found  to  yield  the  greatest  amount  of  nitrogen  in  the  form 
of  monamino  acids.  Approximately  25  per  cent  of  the  nitrogen 
split  off  was  in  the  form  of  amids,  while  the  diamino  nitrogen  has 
been  found  in  still  smaller  amounts,  usually  not  more  than  10  per  cent 
of  the  total  nitrogen  dissolved, 

It  is  not  necessary  to  discuss  in  detail  the  studies  previously  made 
on  this  subject.  It  is  sufficient  to  say  that  too  much  importance  can 
hardly  be  given  to  the  nitrogen  of  soils.  The  element  lies  at  the  very 
foundation  of  plant  growth.  The  use  of  nitrogenous  fertilizers  has 
assumed  enormous  proportions  throughout  the  world.  In  Hawaii 
extremely  heavy  applications  have  been  made  for  many  years,  and 
the  tendency  during  the  past  few  years  has  been  toward  even  greater 
applications.  Many  of  the  soils,  however,  contain  a  relatively  high 
percentage  of  nitrogen.  In  some  instances,  even  where  very  heavy 
applications  of  nitrogenous  fertilizers  are  made,  the  soils  contain  0.5 
per  cent  or  more  of  nitrogen. 

Investigations  on  nitrification  and  ammonification  in  different 
Hawaiian  soils  have  been  under  way  in  this  laboratory  for  some  time, 
and  the  results  obtained  have  been  of  such  nature  as  to  emphasize  the 
need  for  a  better  understanding  of  the  chemical  nature  of  the  nitrogen 
bodies  contained  in  these  soils.  Studies  have  accordingly  been  under- 
taken on  this  subject,  employing  the  process  of  acid  hydrolysis.  In 
this  work  the  nitrogen  as  a  whole  has  been  studied  by  subjecting  to 
hydrolysis  weighed  portions  of  the  original  soils.  On  account  of  the 
great  importance  generally  attached  to  humus,  and  the  limited  state 
of  knowledge  concerning  the  chemistry  of  this  material,  some  study 
was  devoted  to  the  alkali  soluble  nitrogen  bodies. 

Since  the  substances  to  be  investigated  originally  came  from  a  num- 
ber of  plants,  the  protein  of  which  has  not  been  sufficiently  studied, 
and  have  probably  already  undergone  much  change  through  the  action 
of  bacteria,  there  are  no  definitely  understood  materials  to  start  with. 
The  hydrolytic  products  obtained,  therefore,  leave  much  room  for 
speculation.  Just  how  far  the  cleavages  had  already  gone  in  the  soils 
previous  to  treatment  remains  a  matter  for  much  further  work  before 
definite  conclusions  can  be  drawn. 

)  Loc.  cit. 


THE  NITROGEN  OF  HAWAIIAN  SOILS. 

The  soils  used  in  this  investigation  belong  to  the  latcrite  class  com- 
mon to  the  islands.  Sample  No.  379  is  a  silty  loam,  containing  con- 
siderable amounts  of  organic  matter.  It  was  taken  from  old  pasture 
land  in  the  Kula  district  of  Maui,  where  semiarid  conditions  prevail. 

Sample  Xo.  428  is  a  highly  organic  sandy  soil  from  Glenwood,  in 
the  Hilo  district  of  Hawaii,  where  the  rainfall  is  very  heavy,  but  good 
drainage  prevails.  The  sample  came  from  land  recently  cleared  of  a 
tropical  jungle  and  may  be  considered  virgin  fern  forest  soil. 

Sample  No.  447  is  a  silty  loam  from  the  Kunia  district  of  Oahu, 
where  semiarid  conditions  prevail,  and  is  now  being  used  for  pine- 
apples, but  formerly  was  devoted  to  pasture  for  many  years. 

Sample  Xo.  292  taken  from  the  lands  previously  used  for  rice  experi- 
ments by  this  station,  represents  a  loam  soil  containing  considerable 
gravel,  and  has  been  devoted  to  aquatic  agriculture  for  many  years. 

Samples  Xos.  343  and  345  are  each  rice  soils  of  a  silty  character, 
having  been  taken  from  the  Kaneohe  district  on  the  windward  side  of 
Oahu.  These  lands  have  been  devoted  to  continuous  rice  culture  for 
from  30  to  40  years. 

Sample  No.  347  is  a  highly  humus  soil,  taken  from  the  Punuluu  dis- 
trict of  Oahu,  and  likewise  has  been  continuously  cultivated  in  rice 
for  thirty  or  more  years. 

Samples  Nos.  405  and  406  are  silty  soils  taken  from  the  Kalihi  dis- 
trict of  Oahu,  and  have  been  devoted  to  aquatic  agriculture  for  many 
years,  the  former  to  rice  and  the  latter  to  taro. 

NITRATE  AND  AMMONIA. 

It  was  deemed  of  interest  to  determine  the  amounts  of  nitrate  and 
ammonia  present  in  the  soil  prehminary  to  a  study  of  the  organic 
constituents.  Nitrate  was  determined  from  water  solutions,  by  the 
use  of  the  phenol-disulphonic  acid  method,  while  the  ammonia  was 
determined  by  the  direct  distillation  of  separate  portions  of  the  soil 
with  magnesium  oxid.  The  results  calculated  to  the  water-free  basis 
are  given  in  the  following  table: 

Total  nitrate  and  ammonia  nitrogen  in  Hawaiian  soils. 


Total 
nitrogen, 
per  cent. 

Xitrate  nitrogen. 

Ammonia  nitrogen. 

Soil  No. 

Parts  per 
million. 

Per  cent 

of  total 

nitrogen. 

Parts  per 
million. 

Per  cent 

of  total 
nitrogen. 

379 

0.592 
.770 
.  354 
.122 

.220 

1.241 

.  r  '. 

.     10 
4.5 

4 
0 
0 

70 
1 

15 

0. 109 
.  58 1 
1.751 

.328 
.000 
.000 

.050 
.329 

10 
220 
10 
10 
22 
32 
130 
50 
GO 

0.169 

428 

2.  <>1 

447 

292 

343 

1.000 

34") 

1.468 

347 

1.048 

40') 

406 

].  310 

8 

The  above  data  show  that  the  soils  of  Hawaii  are  similar  to  soils 
elsewhere  in  that  the  nitrogen  present  as  nitrate  and  ammonia  consti- 
tutes but  a  small  percentage  of  the  total  nitrogen.  In  contrast  to 
ordinary  soils  the  ammonia  content  in  most  instances  was  considerably 
greater  than  the  nitrate.1  The  nitrogen  of  these  soils  exists,  there- 
fore, very  largely  in  organic  combinations. 

ORGANIC  NITROGEN. 

When  a  study  is  made  of  the  organic  nitrogen  greater  difficulties 
are  at  once  encountered.  The  isolation  of  the  various  individual 
nitrogen  compounds  occurring  in  soils  must  necessarily  be  a  tedious 
undertaking.  It  has  been  shown,  however,  that  by  means  of  the 
hydrolytic  method  as  used  in  the  study  of  protein  chemistry,  some 
conception  can  be  obtained  regarding  the  make-up  of  the  nitrogen 
bodies  of  the  soil.  By  the  use  of  this  method  the  amounts  of  nitrogen 
split  off  in  the  form  of  amids,  diamino,  and  monamino  acids  are 
relatively  easily  determined. 

Partly  on  account  of  the  readiness  with  which  these  determinations 
can  be  made,  and  partly  for  the  reason  that  the  soil  nitrogen  may 
reasonably  be  supposed  to  have  originated  largely  from  vegetable 
proteins,  many  of  which  are  known  to  be  susceptible  to  complete 
hydrolysis,  use  has  been  made  of  the  process  of  hydrolysis  in  this 
work.  In  addition  it  seems  probable  that  the  action  of  bacteria  on 
soil  nitrogen  is  progressive  and  of  a  hydrolytic  nature. 

On  the  other  hand,  the  work  of  Osborne  2  and  others  show  that 
the  hydrolytic  products  vary  widely  with  the  different  proteins  and 
indicate  that  the  results  obtained  in  soil  studies  by  the  use  of 
hydrolytic  agents  must  be  of  the  most  general  nature.  Nevertheless, 
it  is  believed  that  much  valuable  information  can  be  obtained  in  this 
way. 

In  the  work  reported  in  this  bulletin  the  Osborne-Harris  3  modifi- 
cation of  the  Hausmann  4  method,  as  outlined  by  Jodidi 5  in  his 
studies  on  Iowa  soils,  has  been  used.  The  hydrolysis  was  conducted 
by  heating  to  boiling  under  a  reflux  condenser  for  10  hours  50-gram 
portions  of  the  air-dried  soils  with  750  cubic  centimeters  strong  hydro- 
chloric acid,  filtering,  and  making  the  filtrate  to  1  liter.  Aliquots 
of  the  solution  thus  obtained  were  used  for  the  determination  of  the 
amid,  basic,  and  nonbasic  nitrogen.  The  amid  nitrogen,  determined 
as  ammonia  by  the  direct  distillation  of  the  solutions  after  making 
alkaline  with  magnesium  oxid,  would  also  contain  the  ammonia  orig- 

1  The  occurrence  of  large  quantities  of  ammonium  compounds  is  a  phenomenon  common  to  many 
Hawaiian  soils. 

2  The  Vegetable  Proteins.    London  and  New  York,  1909. 
8  Jour.  Amer.  Chem.  Soc.,  25  (1903),  p.  323. 

'Ztschr.  Physiol.  Chem.,  27  (1899),  p.  95. 
» Iowa  Sta.  Research  Bui.  1  (1911). 


9 


inally  present  in  the  soil.     This  was  deducted  from  the  amounts 
found. 

The  basic  nitrogen  was  determined  by  precipitation  with  phospho- 
tungstic  acid  in  water  solutions  obtained  by  filtering  the  residue  left 
after  the  distillation  with  magnesium  oxid.  As  shown  by  previous 
investigators,1  the  nitrogen  thus  obtained  in  this  precipitate  occurs 
largely  in  the  form  of  diamino  acids.  Purins,  alkaloids,  etc.,  may 
also  be  precipitated  by  phosphotungstic  acid,  and,  therefore,  if  pres- 
ent, would  be  contained  in  this  group.  The  nonbasic  nitrogen  was 
determined  by  subtracting  the  sum  of  the  other  two  groups  from  the 
total  nitrogen  dissolved.  While  this  group  is  now  referred  to  by 
some  investigators  as  monamino  acids,  Jodidi  and  Robinson  have 
each  brought  forth  evidence  that  it  is  not  made  up  wholly  of  mon- 
amino acids.  Just  what  other  bodies  occur  in  this  group  is  not  yet 
known.  The  results  obtained,  calculated  as  in  all  other  cases  in 
this  bulletin  on  the  water-free  basis,  are  given  in  the  following  table: 

Organic  nitrogen  of  Hawaiian  soils. 
[Soluble  in  hydrochloric  acid.] 


Per  cent 

of  total 

nitrogen. 

Per  cent  of  the  soil. 

Per  cent  of  nitrogen  in  solution. 

Soil  No. 

Amid. 

Basic 
nitrogen. 

Nonbasic 
nitrogen. 

Total. 

Amid. 

Basic. 

Nonbasic. 

379 

71.96 

72.59 
67.51 
91.80 
90.91 
88.52 
80.42 
84.62 
91.22 

0.095 
.141 
.074 
.024 
.054 
.042 
.225 
.042 
.079 

0.030 
.028 
.033 
.012 
.020 
.017 
.097 
.019 
.055 

0.300 
.368 
.131 
.075 
.124 
.131 
.663 
.099 
.276 

0.426 
.559 
.239 
.112 
.200 
.193 
.998 
.165 
.416 

22.30 
24.79 
30.96 
21.43 
27.00 
21.76 
22.55 
25.45 
18.99 

7.04 
5.01 
13.81 
10.71 
10.00 
8.81 
9.72 
11.51 
13.22 

70.42 

428 

66.27 

447 

54.82 

292 

66.96 

343 

62.00 

345 

67.88 

347 

66.43 

405 

60.00 

406 

66.35 

23.91 

9.98 

64.57 

The  above  table  shows  that  there  is  considerable  variation  in  the 
amounts  of  soluble  nitrogen  in  different  soils.  In  soil  No.  447  only 
67.51  per  cent  of  the  total  nitrogen  was  dissolved,  while  No.  292 
yielded  91.80  per  cent.  Concerning  the  insoluble  nitrogen  very  little 
is  known. 

AMIDS. 

Upon  distilling  the  solutions  after  making  them  alkaline  with  mag- 
nesium oxid  approximately  25  per  cent  of  the  nitrogen  in  solution  was 
obtained  in  the  form  of  ammonia,  here  referred  to  as  amid  nitrogen, 
and  all  the  soils  studied,  with  the  exception  of  Nos.  447,  343,  and  406, 
yielded  approximately  the  same  relative  amounts  of  nitrogen  as 
ammonia.     It  Is  of  interest  to  note  that  the  amids  constitute  a 


33297°— 14- 


1  Jodidi,  loc.  cit.;  Robinson,  loc.  cit. 


10 

considerably  higher  percentage  of  the  nitrogen  of  soils  than  are 
reported  to  occur  in  vegetable  proteins.  For  example,  the  investiga- 
tions of  Osborne  show  that,  on  an  average,  the  seed  proteins  con- 
tained 11.6  per  cent  of  their  nitrogen  as  amids.  In  a  few  cases, 
however,  as,  for  example,  gliadin  from  wheat  and  rye  and  hordein 
from  barley,  the  amids  comprised  more  than  20  per  cent  of  the  total 
nitrogen.  On  the  other  hand,  a  large  number  of  proteins  studied 
were  found  to  contain  considerably  less  than  10  per  cent  of  their 
nitrogen  in  amid  form. 

A  direct  comparison  of  the  amid  nitrogen  of  the  above  soils  with 
that  found  in  soils  elsewhere  is  possible  in  a  few  cases  only,  for  the 
reason  that  the  strengths  of  the  acid,  and  the  lengths  of  the  time  of 
digestion,  used  in  the  investigations  on  this  subject  vary  so  greatly. 
With  the  results  obtained  by  Jodidi,  however,  our  data  are  compar- 
able, and  from  his  work  on  Michigan  peat  and  Iowa  soils  the  amounts 
of  amid  nitrogen  found  were  approximately  the  same  as  those  found 
in  Hawaiian  soils. 

BASIC    NITROGEN. 

The  percentage  of  nitrogen  precipitated  by  phosphotungstic  acid 
was  found  to  vary  considerably  in  the  different  soils  studied,  but 
on  the  average  to  be  similar  to  the  amounts  reported  by  Jodidi.  In 
these  studies  no  attempt  was  made  to  prove  the  nature  of  these  nitrogen 
compounds,  but  from  the  work  of  others  it  seems  permissible  to 
consider  them  as  being  composed  principally  of  diamino  acids.  It  is 
noteworthy  that  the  percentages  of  basic  nitrogen'  in  soils  fall  far 
below  the  percentages  found  in  the  majority  of  vegetable  proteins. 
With  the  exception  of  glutenin  from  wheat,  gliadin  from  wheat  and 
rye,  hordein  from  barley,  and  zein  from  maize,  the  basic  nitrogen 
comprises  more  than  20  per  cent  of  the  total  nitrogen  in  the  vegetable 
proteins  previously  studied,  and  in  a  number  of  instances  even  more 
than  30  per  cent  of  it.  The  basic  nitrogen  compounds  of  Hawaiian 
soils  comprise  only  about  10  per  cent  of  the  total  nitrogen. 

Since  the  principal  diamino  acids  that  occur  in  vegetable  proteins 
are  arginin,  histidin,  and  lysin,  each  of  which  can  be  precipitated 
from  dilute  solutions  by  phosphotungstic  acid,  it  may  be  assumed 
that  these  compounds  contain  the  principal  diamino  nitrogen  split 
off  in  the  hydrolysis  of  soil  organic  matter.  The  amounts  found 
vary  considerably.  This  may  be  accounted  for  in  part  by  the  fact 
that  the  phosphotungstic  acid  method,  in  order  to  give  reliable  results, 
must  be  conducted  under  as  definite  conditions  as  possible.  In  view 
of  the  presence  in  the  solution  of  various  inorganic  salts  dissolved  in 
the  hydrochloric  acid  digestion,  it  is  hardly  to  be  supposed  that  the 
conditions  of  this  precipitate  were  indentical  with  the  different  soils. 
The  precipitate  is  slightly  soluble  in  the  solutions  employed,    and 


11 

at  the  same  time  somewhat  difficult  to  separate  entirely  from  the 
nonbasic  nitrogen  in  the  solution.  For  these  reasons  some  variation 
in  the  results  is  to  be  expected. 

NONBASIC    NITROGEN. 

The  percentages  of  nonbasic  nitrogen,  with  the  exception  of  that 
from  soil  No.  447.  were  found  to  be  remarkably  similar  in  every  in- 
stance, amounting  to  about  two-thirds  of  the  nitrogen  dissolved  by 
boiling  hydrochloric  acid.  In  this  respect  the  soluble  nitrogen  of  soils 
is  quite  similar  to  that  of  vegetable  proteins.  The  nonbasic  nitrogen 
of  soils  is  looked  upon  as  being  composed  largely,  but  not  entirely,  of 
monamino  acids,  and  probably  such  as  are  obtained  in  the  hydrolysis 
of  protein.  Robinson,1  for  example,  isolated  leucin  and  isoleucin 
from  hydrochloric-acid  solutions  of  Michigan  peat.  Doubtless* other 
monamino  acids  occur  in  the  solutions.  It  is  claimed,  however,  that 
a  considerable  portion  of  the  nonbasic  nitrogen  of  soils  occurs  in  forms 
other  than  as  monamino  acids.  Robinson,  by  the  use  of  the  Van  Slyke 
nitrous  acid  method  for  the  determination  of  monamino  acids,  found 
considerably  less  monamino  acid  in  solution  than  was  necessary  to  ac- 
count for  the  nonbasic  group,  while  Jodidi  arrived  at  similar  conclusions 
by  the  use  of  the  formaldehyde  titration  method.  Osborne,2  has  pre- 
sented data  supporting  the  idea  that  the  nonbasic  nitrogen  obtained 
from  vegetable  proteins  actually  occurred  as  monamino  acids.  As 
yet  no  explanation  of  this  difference  between  the  nitrogen  of  soils 
and  that  of  vegetable  proteins  has  been  proposed.  The  soluble  non- 
basic  nitrogen  in  Hawaiian  soils  approximates  the  amounts  found  in 
soils  elsewhere. 

EFFECTS  OF  AERATION  ON  SOIL  NITROGEN. 

Some  references  have  already  been  made  to  the  fact  that  a  wide 
range  in  the  degree  of  aeration  prevails  in  different  Hawaiian  soils, 
and  that  some  of  the  soils  studied  in  this  investigation  represent  ex- 
tremes in  this  respect.  By  reference  to  the  previous  description  of 
the  soils  it  is  seen  that  soils  Nos.  379,  428,  and  447  represent  aerated 
soils,  379  and  447  particularly  so,  since  they  are  taken  from  well 
aerated  land  in  sections  where  semiarid  conditions  have  prevailed 
for  many  years.  The  remaining  soils  studied  represent  anaerobic  con- 
ditions, since  they  have  been  used  in  aquatic  agriculture  a  large  part 
of  the  time  for  many  years.  So  far  as  known  no  nitrogenous  fer- 
tilizers of  any  sort  have  been  applied  to  any  of  these  soils. 

It  is  generally  held  that  the  production  of  ammonia  from  organic 
nitrogen  is  necessary  before  its  nitrification  can  takeplace,  and  that  am- 
monia can  be  formed  by  a  wide  range  of  soil  organisms.    Some  of  these 

i  Loc.  cit.  2  The  Vegetable  Proteins.    London  and  New  York,  1909. 


12 

are  aerobic,  some  anaerobic,  while  still  others  are  able  to  act  under 
either  of  the  two  conditions.  It  has  also  been  shown  at  this  station  1 
that  ammonification  actually  takes  place  in  soils  during  the  time  of 
submergence.  The  relative  amounts  of  amid,  basic,  and  nonbasic 
nitrogen  occurring  in  soils  which  had  long  been  subjected  to  extreme 
conditions  of  aeration  were  determined  to  obtain  evidence  as  to  the 
nature  of  the  chemical  changes  induced  by  the  organisms  when  oper- 
ating under  the  two  sets  of  conditions. 

By  again  referring  to  the  table  (p.  9)  it  will  be  seen  that  the  per- 
centages of  the  total  nitrogen  dissolved  by  hydrochloric  acid  were 
greater  in  every  instance  in  the  soils  long  subjected  to  anaerobic  con- 
ditions. On  an  average  70.69  per  cent  of  the  total  nitrogen  was  dis- 
solved from  aerated  soils,  while  87.93  per  cent  was  rendered  soluble 
in  the  unaerated  soils.  These  data  indicate  that  the  putrefactive 
decay,  which  evidently  predominates  in  submerged  soils,  leaves  the 
nitrogen  in  a  form  more  easily  dissolved  by  hydrochloric  acid  than 
the  process  of  eremacausis,  that  takes  place  under  aerated  conditions. 

The  relative  amounts  of  the  different  groups  obtained  from  the 
soils  representing  the  two  classes  of  conditions,  however,  were  found 
to  be  quite  similar  in  most  instances.  The  table  showing  the  nitrate 
and  ammonia  present  (p.  7)  indicates  that  with  the  exception  of 
soils  Nos.  347  and  406,  those  representing  unaerated  conditions  con- 
tained next  to  no  nitrate.  The  nitrate  found  in  the  remaining  un- 
aerated soils  was  formed  almost  entirely  during  the  time  of  drying 
out  in  the  laboratory.  These  samples  were  taken  from  the  field  in 
a  wet  state  and  then  contained  practically  no  nitrate.  In  fact,  ni- 
trification scarcely  takes  place  at  all  in  submerged  Hawaiian  soils. 
The  data,  therefore,  fail  to  give  any  indication  of  a  fundamental  dif- 
ference in  the  nature  of  the  hydrolyses  which  take  place  under  aerobic 
and  anaerobic  conditions. 

HUMUS  NITROGEN. 

The  alkali  soluble  organic  matter  of  soils,  usually  known  as  humus, 
is  generally  considered  to  be  of  special  importance.  Only  a  part  of 
the  organic  matter  present  in  soils  occurs  as  humus,  and  generally 
very  little  attention  is  paid  to  the  remaining.  For  this  reason  some 
study  has  been  given  to  the  nitrogen  bodies  contained  in  it.  In  this 
investigation  it  was  hoped  to  learn  something  regarding  the  chemical 
make-up  of  these  bodies  by  determining  the  amounts  of  the  different 
nitrogen  groups  actually  present.  Some  light  was  also  sought  on  the 
question  whether  or  not  the  alkali  soluble  nitrogen  bodies  are  really 
different  from  the  organic  nitrogen  of  soils  as  a  whole.     The  soils  used 

i  Hawaii  Sta.  Bui.  31. 


13 

in  this  phase  of  the  work  were  the  same  as  those  employed  in  the 
studies  reported  in  the  preceding  pages. 

NITROGEN    DISSOLVED    IN    THE    PRELIMINARY    1    PER    CENT 
HYDROCHLORIC  ACID  EXTRACTION. 

Calcium  and  magnesium  are  generally  combined  to  some  extent 
with  the  humus  bodies  in  such  a  way  as  to  render  the  organic  matter 
less  soluble  in  dilute  alkalis.  In  order  to  break  up  such  combina- 
tions the  soils  are  treated  with  1  per  cent  hydrochloric  acid  until  no 
further  amounts  of  calcium  and  magnesium  are  dissolved.  It  is 
customary  in  humus  determinations,  then,  to  dissolve  the  humus 
bodies  in  4  per  cent  ammonia  solution.  In  brief  investigations  l 
carried  on  in  this  laboratory  it  was  observed  that  the  dilute  hydro- 
chloric acid  extracts  obtained  in  the  preliminary  treatment  contained 
considerable  organic  matter.  In  one  instance  the  solution  was 
darkly  colored  and  found  to  contain  a  notable  amount  of  organic 
matter.  Usually  such  solutions  are  discarded.  It  has  been  shown, 
however,  that  considerable  amounts  of  nitrogen  are  dissolved  from 
certain  soils  2  in  this  preliminary  acid  extraction.  In  the  work  here 
reported  the  soils  were  first  extracted  with  1  per  cent  hydrochloric 
acid,  then  filtered  and  washed  to  neutrality.  The  solutions  thus 
obtained  should  contain  the  ammonia  originally  present.  The  solu- 
tions were  evaporated  to  a  small  volume  and  the  nitrogen  in  them 
was  determined  by  the  Kjeldahl  method,  with  the  following  results: 

Nitrogen  of  soils  soluble  in  cold  1  per  cent  hydrochloric  acid. 


Soil  No. 

Per  cent 
of  soil. 

Per  cent 
of  total 
nitrogen. 

Soil  No. 

Per  cent 
of  soil. 

Per  cent 
of  total 
nitrogen. 

379 

0.019 
.041 
.012 
.004 
.004 

3.21 
5.33 
3.39 
3.28 
1.82 

345 

0.007 
.029 
.009 
.012 

3.21 

428 

347 

2.34 

447 

[  405... 

4.61 

292 

'  406... 

2.63 

343 

By  comparing  these  data  with  those  given  in  the  first  table  it  will 
be  seen  that  in  every  instance  the  soils  contained  only  about  one-half 
as  much  ammonia  nitrogen  as  was  dissolved  by  1  per  cent  hydrochloric 
acid,  while  in  a  number  of  instances  still  greater  amounts  of  nitrogen 
were  dissolved.  Some  organic  nitrogen,  therefore,  was  thus  dis- 
solved, although  the  amounts  were  small. 

In  preparing  the  humus  solutions  for  studies  on  the  nitrogen 
bodies  a  3  per  cent  solution  of  sodium  hydrate  was  employed.  With 
Hawaiian  soils  sodium  hydrate  solution  has  a  special  advantage  of 

i  Hawaii  Sta.  Press  Bui.  33. 

2  Rimbach,  Jour.  Amer.  Chem.  Soc.,  22  (1900),  p.  695. 


14 

causing  much  less  deflocculation  of  the  clay,  so  that  by  ordinary 
filtration  the  solutions  can  be  freed  from  all  but  traces  of  clay.  Forty 
grams,  after  extracting  with  dilute  hydrochloric  acid,  were  treated 
with  2,000  cubic  centimeters  of  3  per  cent  sodium  hydrate  solution 
for  a  period  of  two  days,  with  occasional  shaking  during  the  first  day. 
The  solutions  were  siphoned  off  and  aliquot  portions  used  in  the 
studies. 

SEPARATION  OF  DIFFERENT  FORMS  OF  NITROGEN  IN  HTJMTJS. 

A  part  of  the  humus  can  be  precipitated  from  the  alkali  solutions 
by  acids,  and  this  method  has  been  used  for  obtaining  so-called  pure 
humus.  The  amounts  precipitated,  however,  vary  with  the  amount 
of  acid  used.  Shorey  *  has  shown  that  after  filtering  out  the  pre- 
cipitate obtained  by  acidifying  the  humus  solution  a  still  further 
precipitate  can  be  obtained  by  carefully  neutralizing  the  filtrate, 
and  that  of  the  precipitates  thus  obtained  each  contains  nitrogen. 
The  humus  extract  made  with  a  2  per  cent  sodium  hydrate  solution 
was  found  to  contain  0.0399  gram  nitrogen  per  100  cubic  centimeters 
of  solution.  The  hydrochloric  acid  filtrate  he  found  to  contain  0.0251 
gram  of  nitrogen  per  100  cubic  centimeters  of  the  original  solution, 
and  on  neutralizing  this  filtrate  with  caustic  soda  the  precipitate 
formed  was  found  to  contain  0.0168  gram  of  nitrogen. 

In  the  work  here  reported  hydrochloric  acid  was  carefully  added 
to  1,000  cubic  centimeter  portions  of  the  humus  solution  (corre- 
sponding to  20  grams  of  soil)  to  apparent  neutrality  to  litmus  paper, 
then  20  cubic  centimeters  of  1  per  cent  hydrochloric  acid  was  added, 
the  precipitate  formed  was  collected  on  a  filter  and  washed.  In  this 
way  the  humus  matter  was  roughly  separated  into  two  parts.  The 
precipitates  thus  obtained  were  afterwards  subjected  to  acid  hydrol- 
ysis by  boiling  with  400  cubic  centimeters  strong  hydrochloric 
acid  for  a  period  of  10  hours,  and  then  filtering  and  washing  the 
residue.  The  amid  and  basic  nitrogen  contained  in  the  original 
humus  solutions,  in  the  filtrates  obtained  from  precipitating  the 
solutions  with  dilute  hydrochloric  acid,  and  in  those  obtained  upon 
hydrolyzing  the  humus  precipitates,  have  been  determined. 

The  following  table  shows  the  total  nitrogen  contained  in  the 
original  humus  solutions,  and  that  in  the  portions  obtained  by  the 
various  separations: 

i  Hawaii  Sta.  Rpt.  1906. 


15 

The  nitrogen  of  humus. 

Humus  nitrogen 

Ilumus  numus  nitrogen 

precipitated  by 

Humus 

Total 

Total 

humus 

nitro- 

precipitated by 

Total 

HC1. 

nitro- 

humus 

Humus 

gen  not 

HC1. 

nitro- 
,gen  in 

soil. 

Per 
cent  of 

soil. 

gen 
not 
precipi- 
tated 

nitro- 
gen by 

addi- 
tion 

gen 
deter- 
mined 

di- 
rectly. 

Per 
cent  of 

soil. 

nitro- 
gen in 

per 
cent  of 

precipi- 
tated 
byHCl. 
Per 

Soil  No. 

Non- 
Hydro-  hydro* 

Hydro- 
lyzable. 

Non- 

hydro- 

Lysable. 

Per 

lyzable  lyzable 

byHCl. 

(a+b+ 

soil 

cent  of 

Per 

(a).  Per  (b).  Per 

(c).  Per 

c).    Per 

nitro- 

total 

cent  of 

cent  of 
soil. 

cent  of 
soil. 

cent  of 
soil. 

cent  of 
soil. 

gen. 

humus 
nitro- 

humus 
nitro- 

humus 
nitro- 

gen. 

gen. 

gen. 

347 

1.241 

0.362 

0.125 

0.315 

0.802 

0.774 

62.37 

39.28 

4.").  14 

15.59 

428 

.770 

.289 

.051 

.247 

.587 

.590 

76.62 

42.08 

49.23 

8.69 

379 

.592 

.270 

.041 

.129 

.440 

.439 

71.  16 

29.32 

61.36 

9.32 

406 

.456 

.100 

.027 

.117 

.244 

.226 

49.56 

47.96 

40.98 

11.06 

447 

.354 

.094 

.026 

.105 

.225 

.215 

60.79 

46.67 

41.78 

11.56 

345 

.218 

.066 

.019 

.070 

.147 

67.43 

45.16 

42.58 

12.26 

343 

.220 

.061 

.012 

.069 

.142 

.127 

57.73 

48.59 

42.96 

8.45 

405 

.195 

.044 

.012 

.067 

.123 

.123 

63.08 

54.47 

35.  77 

9.76 

292 

.122 

.031 

.012 

.015 

.058 

.058 

67.54 

25.86 

53.45 

20.69 

Average 

64.36 

42. 15 

45.92 

11.93 

1 

The  above  data  show  that  the  humus  nitrogen  varied  with  different 
soils,  but  averaged  64.36  per  cent  of  the  total  nitrogen.  In  every 
instance,  except  two,  more  than  one-half  of  the  nitrogen  was  dissolved 
by  dilute  alkali,  while  in  two  instances  practically  three-fourths  of  it 
was  thus  extracted.  The  bodies  precipitated  with  dilute  hydro- 
chloric acid  also  contained  nitrogen  in  varying  amounts.  The  nitro- 
gen bodies  precipitated  by  hydrochloric  acid  upon  subsequent 
hydrolysis  yielded  by  far  the  greater  portion  of  their  nitrogen  to  the 
solutions,  the  insoluble  residues  having  been  found  to  contain  11.93 
per  cent  of  the  humus  nitrogen.  By  these  methods,  therefore,  the 
nitrogen  of  soils  can  be  separated  into  fractional  parts. 

AMID    XITROGEX. 


The  amid  nitrogen  in  the  original  humus  solutions  was  first  deter- 
mined by  evaporating  the  solutions  on  the  water  bath,  after  slightly 
acidifying  with  hydrochloric  acid,  then  making  alkaline  with  mag- 
nesium oxid  and  distilling.  The  relatively  high  percentages  of 
ammonia  thus  obtained  suggested  that  some  hydrolysis  had  taken 
place  during  the  time  of  the  evaporation  on  the  water  bath,  possibly 
through  the  action  of  the  hydrochloric  acid  present.  In  order  to 
eliminate  this  possibility,  separate  portions  were  distilled  directly 
with  magnesium  oxid,  after  having  been  slightly  acidified  with 
hydrochloric  acid.  In  like  manner  the  solutions,  obtained  after 
filtering  out  the  humus  matter  precipitated  by  hydrochloric  acid, 
were  distilled  with  magnesium  oxid,  and  also  the  solutions  obtained 


16 

by  hydrolyzing  the  humus  precipitate.     The  results  are  shown  in  the 
following  table: 

Amid  nitrogen  in  humus. 


Solutions 

slightly 

acidified 

and 

evaporated 

before 

distillation. 

Per  cent 

of  soil. 

Solutions 
slightly 

acidified. 

No  evapo- 
ration. 

Per  cent 
of  soil. 

Not  pre- 
cipitated by 
HCl.  (a). 
Per  cent 
of  soil. 

Precipi- 
tated by 
HCl.  (b\ 
Per  cent 

of  soil. 

Total. 

Soil  No. 

a+b. 

Per  cent 
of  total 

soil 
nitrogen. 

Per  cent 

of 

humus 

nitrogen. 

317 

0.100 
.073 
.059 
.039 
.033 
.025 
.020 
.026 
.009 

0.087 
.073 
.052 
.028 
.026 
.018 
.020 
.017 
.012 

0.095 
.074 
.056 
.040 
.031 
.022 
.025 
.014 
.008 

0.073 

.056 
.061 
.020 
.027 
.016 
.018 
.023 
.011 

0.168 
.130 
.117 
.060 
.058 
.038 
.043 
.037 
.019 

13.54 
16.88 
19.76 
13.15 
16.38 
17.43 
19.54 
18.97 
15.57 

21.70 

428 

22.03 

379 

26.65 

406 

26.55 

447 

26.98 

345 

25.85 

343 

33.86 

405 

42.53 

292 

32.76 

16.80 

28.77 

The  relatively  high  percentages  of  amid  nitrogen  obtained  from  the 
original  humus  solutions  is  noteworthy,  as  is  also  the  fact  that  the 
nitrogen  bodies  not  precipitated  by  hydrochloric  acid  contained 
practically  all  of  the  amid  nitrogen  existing  as  such  in  the  humus 
solution.  On  an  average,  the  hydrochloric  acid  precipitate  yielded 
upon  hydrolysis  practically  the  same  amounts  of  amid  nitrogen  as 
were  contained  in  the  original  humus  solutions.  The  total  amid 
nitrogen  contained  in  the  humus,  when  calculated  to  the  percentages 
of  the  total  soil  nitrogen,  presents  some  variation,  and  on  the  average 
amounted  to  16.80  per  cent  of  the  total  soil  nitrogen.  When  such 
data  were  calculated  to  the  basis  of  the  humus  nitrogen  it  was  found 
that  the  relative  amounts  of  amid  nitrogen  contained  increased  with 
a  decrease  in  the  humus  nitrogen.  In  other  words,  relatively  greater 
amounts  of  amids  occurred  in  soils  which  contain  a  low  percentage  of 
humus  nitrogen.  This  may  be  purely  a  coincidence,  but  is  probably 
due  to  the  fact  that  either  in  the  preparation  of  the  humus  solution, 
or  in  the  process  of  determining  the  amid  nitrogen  in  it,  a  certain 
amount  of  hydrolysis  took  place  which  would  tend  to  markedly 
increase  the  relative  amounts  of  amid  obtained  from  those  humus 
solutions  containing  the  smallest  amounts  of  nitrogen.  On  an 
average,  28.77  per  cent  of  the  humus  nitrogen  was  found  to  be  present 
as  amids.  It  will  be  recalled  that  the  amid  nitrogen  obtained  upon 
hydrolyzing  the  soils  as  a  whole  amounted  to  considerably  smaller 
percentages  of  the  nitrogen  dissolved. 

BASIC    NITROGEN. 

The  basic  nitrogen  bodies  in  humus  were  determined  by  the  phos- 
photungstic  acid  method,  as  already  outlined.  The  results  are  re- 
corded in  the  following  table: 


17 


Basic  nitrogen  in  humus. 


In  the  orig- 
inal humus 
solution. 
Per  cent  of 
soil. 

Not  pre- 
cipitated 
by  HCl. 
(a). 
Per  cent  of 
soil. 

Precipita- 
ted l)v  11(1. 

(b). 

Per  cent  of 

soil. 

Total. 

Soil  No. 

a  +  b. 

Percent  of 

soil. 

Percent  of 
total  soil 
nitrogen. 

Per  cent  of 

total  humus 
nitrogen. 

347             

0.028 
.027 
.017 
.015 
.021 
1.003 
1.004 
.011 
.014 

0.024 
.025 
.021 
.014 
.019 
.017 
.017 
.009 
.009 

0.010 
.010 
.007 
.009 
.006 
.017 
.008 
.008 
.017 

0.034 
.035 

.028 
.023 
.025 
.034 
.025 
.017 
.026 

2.  7  4 
4.  SI 
4.73 
5.05 
7.06 
16.59 
11.36 
8.71 
21.31 

4.39 

428 

5.93 

379 

6.88 

406 

Ki.  L8 

447 

LI.  63 

345 

23. 1  > 

343 

19.68 

405 

19.54 

292 

44.83 

1  There  is  apparently  some  error  in  these  determinations. 

It  was  found  that  the  basic  nitrogen  amounted  to  practically  the 
same  percentages  when  determined  directly  from  the  original  humus 
solutions  as  were  present  in  the  filtrates  from  the  hydrochloric  acid 
precipitate.  The  amounts  of  basic  nitrogen  split  off  in  the  hydroly- 
sis of  the  precipitates  were  relatively  smaller,  as  compared  with  the 
amid  nitrogen,  than  were  obtained  in  the  filtrates.  The  total  basic 
nitrogen,  calculated  to  percentages  of  the  total  soil  nitrogen,  in- 
creased with  a  decrease  in  the  amount  of  nitrogen  in  the  soil,  and 
when  calculated  to  percentages  of  the  total  humus  nitrogen  the 
same  relationships  are  even  more  marked.  Soil  No.  347,  containing 
0.77  per  cent  humus  nitrogen,  yielded  only  4.39  per  cent  of  it  as 
basic  nitrogen,  while  soil  No.  292,  containing  0.058  per  cent  humus 
nitrogen,  yielded  44.83  per  cent  of  it  as  basic  nitrogen. 

In  view  of  the  analytical  error  involved  in  the  determination  of  the 
basic  nitrogen,  it  is  unsafe  to  generalize  concerning  the  relatively 
great  increases  in  the  basic  nitrogen  of  humus  in  passing  from  soils 
with  large  to  soils  of  smaller  humus  nitrogen  content.  It  seems 
probable,  however,  that  hydrolysis  took  place  during  the  alkali  ex- 
traction process. 

XOXBASIC    XITROGEX. 

It  is  obviously  not  permissible  to  consider  the  difference  between 
the  total  nitrogen  in  humus  and  the  amounts  of  amid  and  basic 
nitrogen  that  occur  in  the  original  humus  solutions  as  nonbasic 
nitrogen,  for  the  reason  that  these  solutions  can  not  be  considered  as 
having  been  completely  hydrolyzed.  It  is  well  known,  for  example, 
that  various  proteins  are  quite  soluble  in  alkalis  without  the  proteins 
undergoing  any  particular  hydrolysis,  as  they  can  be  precipitated, 
in  a  more  or  less  unaltered  condition,  from  such  solutions  by  the 
addition  of  acid.  On  the  other  hand,  the  nitrogen  compounds  in  the 
filtrates  obtained  from  the  humus  precipitated  by  dilute  hydrochloric 


18 


acid,  and  also  those  split  off  in  the  hydrolysis  of  the  humus  precipitate, 
may  reasonably  be  considered  as  being  made  up  of  amid,  basic,  and 
nonbasic  nitrogen  compounds.  We  have,  therefore,  calculated  the 
amounts  of  nonbasic  nitrogen  in  these  portions  of  humus.  The 
results  are  shown  in  the  following  table: 

Nonbasic  nitrogen  in  humus. 


In  filtrates  from  HC1 
precipitates. 

In  hydrolyzed  humus 
precipitates. 

Total. 

Soil  No. 

Per  cent 
of  soil. 

Per  cent 
of  nitro- 
gen in 
filtrate. 

Per  cent 
of  humus 
nitrogen. 

Per  cent 
of  soil. 

Per  cent 
of  nitro- 
gen in 
solution. 

Per  cent 
of  humus 
nitrogen. 

Per  cent 
of  soil. 

Per  cent 
of  humus 
nitrogen. 

347 

0.196 
.148 
.052 
.063 
.055 
.031 
.027 
.046 

62.22 
59.92 
40.31 
53. 84 
52.38 
44.28 
39.13 
68.65 

25.32 
25.08 
11.84 
27.87 
25.58 
21.09 
21.26 
37.39 

0.249 
.213 
.203 
.071 
.061 
.033 
.035 
.011 
.003 

68.78 
73.70 
75.18 
71.00 
64.89 
50.00 
57.37 
125.00 
19.67 

32.17 

36.10 
46.24 
30.97 
28.36 
22.45 
27.56 
18.78 
15.18 

0.445 
.361 
.255 
.134 
.116 
.064 
.062 
.057 

57.49 

428 

61.18 

379 

58.08 

406 

58.84 

447 

53.94 

345 

43.54 

343 

48.82 

405 

45.17 

292 

52.59 

25.05 

64.84 

31.98 

53.38 

i  Not  included  in  averages. 

These  data  show  the  relatively  large  amounts  of  nonbasic  nitrogen 
contained  in  humus.  On  the  average  about  25  per  cent  of  the  humus 
nitrogen  occurred  in  the  original  humus  solutions  as  nonbasic  nitrogen 
compounds,  or  52.59  per  cent  when  calculated  to  the  percentage  of 
humus  nitrogen  soluble  in  dilute  hydrochloric  aci'd.  The  solutions 
obtained  upon  hydrolyzing  the  humus  precipitated  by  dilute  hydro- 
chloric acid  yielded  a  still  greater  amount  of  nonbasic  nitrogen. 
On  an  average  64.84  per  cent  of  this  nitrogen  occurred  as  nonbasic, 
which,  when  calculated  to  percentages  of  the  total  humus  nitrogen, 
amounts  to  31.98  per  cent.  By  adding  the  nonbasic  nitrogen  in 
these  two  portions  of  humus  it  is  found  that  53.38  per  cent  of  the 
humus  nitrogen  is  made  up  of  nonbasic  nitrogen  compounds.  By 
referring  to  the  data  previously  presented  (p.  9)  it  will  be  seen  that 
the  relative  amounts  of  nonbasic  nitrogen  in  humus  are  somewhat 
less  than  the  amounts  of  nonbasic  nitrogen  obtained  in  the  hydrolysis 
of  the  soil  as  a  whole. 

Considering  the  different  groups  of  nitrogen  compounds  as  obtained 
from  the  different  portions  of  humus,  the  preceding  data  show  that  the 
humus  contained  slightly  less  amid,  basic,  and  nonbasic  nitrogen  than 
were  split  off  upon  hydrolyzing  the  soil  nitrogen  as  a  whole,  but,  on 
the  other  hand,  the  humus  nitrogen  bodies  as  such  are  made  up  of 
relatively  more  amid  and  basic  nitrogen  than  the  soil  nitrogen  as  a 
whole.  In  other  words,  the  nitrogen  of  soils  soluble  in  3  per  cent 
sodium  hydrate  is  bound  up  in  bodies  differing  somewhat  from  the 


19 

nitrogen  bodies  not  soluble  in  the  solvent.  In  view  of  the  relatively 
large  amounts  of  amid,  basic,  and  nonbasic  nitrogen  contained  in  the 
original  humus  solutions,  it  is  believed  that  considerable  hydrolysis  of 
the  proteins  occurring  in  soils  has  taken  place  through  the  action 
of  bacteria,  and  that  the  humus  nitrogen  is  probably  of  more  immedi- 
ate value  as  a  source  of  available  nitrogen  than  is  the  nonhumus 
nitrogen.  It  seems  justifiable  to  believe,  therefore,  that  the  humiilca- 
tion  process  is  really  one  of  importance  in  soils  as  a  step  toward  the 
production  of  available  nitrogen  compounds. 

DETERMINATION  OF  HUMUS  NITROGEN. 

In  view  of  the  large  amounts  of  amid  nitrogen  obtained  in  the 
original  humus  solutions  some  study  was  directed  to  the  question  of 
methods  for  the  determination  of  total  humus  nitrogen.  The  data 
previously  submitted,  showing  the  total  humus  nitrogen,  were  ob- 
tained by  first  evaporating  the  solutions  after  acidification  with  hy- 
drochloric acid,  then  subjecting  the  residues  to  nitrogen  determination 
by  the  use  of  the  regular  Kjeldahl  method.  The  determination  of 
humus  nitrogen  is  frequently  made  from  ammonia  solutions  of  humus 
after  expelling  the  free  and  combined  ammonia  present  by  distilling 
the  magnesium  oxid.1  But  on  account  of  the  relatively  large  amounts 
of  amid  nitrogen  found  in  the  sodium  hydrate  solutions,  which,  if 
present  in  corresponding  amounts  in  ammonia  solutions  of  humus, 
would  be  lost  in  the  magnesium  oxid  distillation,  there  is  brought 
into  comparison  the  nitrogen  of  these  soils  as  found  in  both  the  sodium 
hydrate  and  ammonia  solutions.  The  results  are  recorded  in  the 
following  table: 

Total  humus  nitrogen  by  different  methods. 


Soil  No. 

Amid 
nitrogen 
in  NaOH 
solution. 
Per  cent 

of  soil. 

Total 
humus 
nitrogen 
in  NaOH 
solution. 
Per  cent 

of  soil. 

Total 
humus 

nitrogen 
in  am- 
monia 

solution. 

Per  cent 
of  soil 

Nitrogen 
absorbed 
from  am- 
monia 
solutions. 
Per  cent 
of  soil. 

Soil  No. 

Amid 
nitrogen 
in  NaOH 
solution. 
Per  cent 

of  soil. 

Total 
humus 
nitrogen 
in  NaOH 
solution. 
Per  cent 
of  soil. 

Total 
humus 

nitrogen 
in  am- 
monia 

solution. 

Per  cent 
of  soil. 

Nitrogen 
absorbed 
from  am- 
monia 
solutions. 
Per  cent 
of  soil. 

347 

428 

379 

406 

447 

0.100 
.073 
.059 
.039 
.033 

0.774 
.590 
.439 
.226 
.215 

0.657 
.609 

.284 
.179 
.218 

0.502 
.753 
.523 
.264 
.147 

345 

!  343 

1  405 

292 

0.025 
.020 
.026 
.009 

0.147 
.127 
.087 
.058 

0.140 
.117 

.099 
.067 

0.085- 
.175 
.139 

.072 

In  some  instances  much  higher  percentages  of  nitrogen  were  found 
in  the  sodium  hydrate  solutions  than  in  the  ammonia  solutions,  and 
in  some  instances  this  difference  about  equals  the  amid  nitrogen  con- 

1  In  determining  the  nitrogen  in  the  ammonia  Folut  ions  of  humus  it  was  found  advantageous  to  evaporate 
to  dryness  two  portions  of  the  solution.  In  one  the  combined  ammonia  only  was  determined,  which 
amounts  were  subtracted  from  the  total  nitrogen  found  in  the  other  without  distilling  with  MgO. 


20 

tained  in  the  sodium  hydrate  solutions.  Rimbach  l  has  shown  that 
the  humus  nitrogen  as  determined  in  sodium  hydrate  and  ammonia 
solutions,  respectively,  occurs  in  greater  amounts  in  the  former  solu- 
tions, from  which  he  concluded  that  sodium  hydrate  dissolves  more 
nitrogen  than  ammonia.  Similar  observations  have  also  been  made 
by  others.  From  work  done  in  this  laboratory  it  is  doubtful  whether 
sodium  hydrate  actually  dissolves  more  nitrogen  than  ammonia,  but 
rather  that  the  increased  amounts  found  represent  amid  nitrogen, 
which  is  lost  in  the  methods  employed  in  the  determination  of  total 
humus  nitrogen  from  ammonia  solutions. 

While  the  amounts  of  ammonia  absorbed  by  the  residues  left  after 
evaporating  to  dryness  ammonia  solutions  of  humus  bear  no  definite 
relation  to  the  amounts  of  humus  nitrogen  present,  the  absorption  of 
ammonia  by  humus  took  place  to  a  considerable  extent,  and  correc- 
tion should  be  made  for  this  in  humus  determinations,  as  has  been 
pointed  out  by  Emery  -  and  others. 

PERCENTAGE  OF  NITROGEN  IN  HTJMTJS  OF  HAWAIIAN  SOILS. 

Hilgard  3  has  shown  that  humus  from  arid  regions  contains  a  higher 
percentage  of  nitrogen  than  humus  from  humid  sections.  He  found 
that  humus  from  arid  regions  contained  on  the  average  15.23  per 
cent  nitrogen,  while  the  humus  from  humid  regions  contained  only 
4.23  per  cent.  In  the  work  on  soils  at  this  station  many  humus  and 
humus  nitrogen  determinations  have  been  made  and  some  of  the 
results  obtained  are  submitted  in  the  following  table: 

Total  humus  arid  humus  nitrogen  in  Hawaiian  soils. 


Soil  "To. 


Humus. 
Fer  rent 
of  soil. 


347 14.81 

423 14.31 

379 8.20 

406 3.0> 

447 3.61 

345 1.74 

343 2.45 

405 1.74 

292 1.80 

282 2.77 

283 1.78 

284 2.72 


Humus 

Humus 

ash.   Per 

nitrogen. 

cent  of 

Per  cent 

soil. 

of  humus. 

2.28 

5.16 

3.89 

4.12 

1.91 

5.35 

1.64 

7.33 

1.54 

5.94 

1.13 

6.88 

1.73 

5.21 

1.07 

4.91  j 

1.87 

3.21 

1.04 

7.87 

.79 

6.40 

.70 

5.62 

285. 

2v6. 
287. 
2S8. 
312. 
313. 
314. 
315. 
316. 
317. 


Soil  No. 


Average. 


Humus. 
Per  cent 
of  soil. 


2.03 
3.15 
3.64 
3.06 
5.26 
4.14 
3.94 
4.33 
3.39 
5.12 


Humus 

ash.   Per 

cent  of 

soil. 


Humus 
nitrogen. 
Per  cent 
of  humus. 


0.63 

.94 

1.38 

1.48 

1.19 

1.44 

1.17 

.79 

.81 

1.18 


9.85 
10.35 
7.31 
5.88 
5.27 
6. 85 
6.81 
6.36 
6.22 
6.32 


5.88 


These  data  show  that  the  humus  of  Hawaiian  soils  contains  nitrogen 
in  amounts  similar  to  those  of  humid  soils  elsewhere.  Some  of  the 
soils  used  in  this  investigation  (Xos.  379  and  447)  came  from  sections 
which  have  been  designated  as  arid,  but  the  arid  conditions  which 
now  prevail  in  these  sections  have  probably  not  existed  as  such  for 


:  Loo.  eft. 

2  Jour.  Amer.  Chem.  Soc.,  22  (1900),  p.  285. 


3  Soils.    New  York  and  London,  1907,  pp.  136, 137. 


21 

many  generations,  perhaps  not  more  than  7'  3,  and  the  humus 

has  been  formed  largely  under  humid  or  semihumid  conditio 

It  is  probable  that  oxidation  takes  place  more  actively  in  arid  than 
in  humid  soils,  which  oxidation  probably  results  hi  a  greater  degi  • 
decomposition  of  the  nonnitrogen  constituents,  thus  leaving  a  humus 
residue  richer  in  nitrogen.  Also,  the  humus  may  be  considered  as 
being  older  than  that  occurring  in  humid  soils,  for  the  reason  that 
greater  amounts  of  plant  residues  are  continually  becoming  incorpo- 
rated with  the  soils  under  conditions  that  are  more  favorable  for  plant 
growth,  such  as  are  offered  by  a  more  abundant  moisture  supply. 
For  these  reasons  (perhaps  others)  it  is  to  be  expected  that  the  humus 
of  arid  soils  would  be  more  largely  composed  of  nitrogen  constituents 
than  that  of  humid  soils. 

STTMMAKY. 

(1)  The  nitrate  and  ammonia  content  of  Hawaiian  soils  constitutes 
only  a  small  percentage  of  the  total  soil  nitrogen. 

(2)  Upon  boiling  different  soils  with  strong  hydrochloric  acid,  the 
amounts  of  nitrogen  dissolved  ranged  from  67.51  per  cent  to  91. 8S  per 
cent  of  the  total  nitrogen.  With  two  exceptions,  the  relative  per- 
centages of  amid  nitrogen,  split  off  in  the  hydrolysis,  were  approxi- 
mately the  same,  amounting  on  the  average  to  23.91  per  cent  of  the 
nitrogen  dissolved.  Basic  nitrogen  occurred  in  the  solutions  in  vari- 
able amounts,  the  average  being  9.98  per  cent  of  the  soluble  nitrogen. 
The  percentages  of  nonbasic  nitrogen,  determined  by  difference, 
proved  to  be  quite  concordant  in  most  of  the  soils,  amounting  on  the 
average  to  64.57  per  cent  of  the  soluble  nitrogen. 

(3)  The  relative  percentages  of  amid  and  basic  nitrogen,  split  off 
in  the  hydrolysis  of  Hawaiian  soils,  stand  in  the  reverse  order  to  that 
in  which  they  occur  in  the  vegetable  proteins;  while  the  percentage  of 
nonbasic  nitrogen  practically  equals  that  found  in  the  vegetable  pro- 
teins. It  had  been  suggested  that  soil  bacteria  attack  the  nitrogen 
bodies  in  such  way  as  to  split  off  the  basic  nitrogen  compounds,  and 
that  these  then  become  ammonified,  or  otherwise  lose  their  identity 
as  di amino  acid  compounds,  possibly  being  partially  converted  into 
amid  forms. 

(4)  Anaerobic  conditions  predominate  in  Hawaiian  soils,  and  under 
such  conditions  the  nitrogen  is  more  soluble  than  in  well  aerated  - 
but  the  relative  percentages  of  the  different  groups  of  organic  nitr  _ 
compounds  seemed  not  to  be  affected  by  the  predominance  of  ofl 
the  other  of  these  conditions. 

(5)  The  amount  of  nitrogen  soluble  in  1  per  cent  hydrochloric  acid 
was  about  twice  a-  large  a-  that  of  amnion:;  3  ally  occurring  in 
the  soil-. 


22 

(6)  The  solubility  in  3  per  cent  sodium  hydrate  varied  from  49.56 
per  cent  to  76.62  per  cent  of  the  total  nitrogen.  Of  the  nitrogen  thus 
dissolved,  57.85  per  cent  was  precipitated  by  dilute  hydrochloric  acid, 
of  which  11.93  per  cent  (expressed  in  percentage  of  the  humus  nitro- 
gen) remained  insoluble  after  boiling  in  strong  hydrochloric  acid  for 
10  hours.  Amids  comprised  28.77  per  cent  of  the  humus  nitrogen,  of 
which  about  one-half  existed  as  amid  in  the  original  humus  solutions, 

,  and  which  remained  in  solution  upon  acidifying  with  hydrochloric  acid. 

The  remaining  half  was  split  off  when  the  humus,  precipitated  by 
hydrochloric  acid,  was  subjected  to  acid  hydrolysis.  The  basic  nitro- 
gen ranged  from  4.39  per  cent  to  44.83  per  cent  of  the  humus  nitrogen, 
increasing  as  the  total  nitrogen  of  the  humus  decreased.  Nonbasic 
nitrogen  was  found  to  constitute  53.38  per  cent  of  the  humus  nitrogen, 
of  which  25.05  per  cent  existed  as  such  in  the  original  humus  solutions. 

(7)  The  amounts  of  amid  and  basic  nitrogen  in  humus  expressed  as 
percentages  of  the  humus  nitrogen  were  found  to  be  higher  than  the 
amounts  obtained  by  subjecting  the  original  soil  to  hydrolysis. 

(8)  In  view  of  the  large  amounts  of  amid  occurring  in  humus  solu- 
tions, it  was  found  better  to  use  sodium  hydrate  as  the  solvent  for 
extracting  humus  that  is  to  be  used  for  total  humus  nitrogen  deter- 
minations. 

(9)  The  humus  of  Hawaiian  soils  contains  a  small  percentage  of 
nitrogen  (5.88  per  cent  as  an  average  of  22  samples),  in  which  respect 
the  humus  of  these  soils  closely  resembles  that  found  in  humid  soils 
in  the  States. 

o 


.. 


UNIVERSITY  OF  FLORIDA 

lllflllilWllllll 

3  1262  08929  1065 


